The ribosome, as the site where genetic information is translated into polypeptides which subsequently fold into proteins, and as the target site for antibiotic action, is an organelle with eminent public health relevance. X-ray crystallography and cryo-electron microscopy (cryo-EM) are the main tools to probe ribosomal structure. RNA has emerged as the most important component of the ribosome, acting as a structural scaffold and promoting most of the functionally important interactions with mRNA and tRNAs, as well as the peptidyl moiety. Understanding the dynamics of RNA in these interactions will be key to the understanding of ribosomal function. Now that atomic structures of ribosomal subunits of prokaryotes are available, the exploration of RNA dynamics can be approached by a combination of cryo-EM of functional states, flexible fitting of X-ray structures, and molecular modeling and molecular dynamic simulations. Pioneering contributions by this lab in the study of ribosome structure and function using cryo-EM, some of which were made in pursuing the goals of the previous funding period of this grant, position us well to take on some of these challenges. This renewal proposal describes a four-year research plan in collaboration with leading experts in ribosome biochemistry, molecular modeling, flexible fitting, and molecular dynamics. The Specific Aims are centered at the exploration of key events of decoding and translocation, recorded as three-dimensional snapshots by cryo-EM, and the inference of the conformational dynamics of the ribosome, and the dynamics of ribosome-ligand interactions from such snapshots. For both decoding and translocation, additional transition states will be trapped for cryo-EM visualization. Attempts will be made to obtain these maps, as well as the density maps of existing states, with resolutions in the range of 7 A to provide more stringent constraints in atomic modeling. We believe that the interdisciplinary efforts proposed are likely to advance the understanding of the molecular basis of translation.